OBJECTIVE: To investigate the 16S rRNA methylase genes and Aminoglycoside modifying enzymes (AMEs) genes in Enterobacter cloacae isolated from the People's Liberation Army 98th Hospital, Huzhou district, Zhejiang province, China. METHODS: 40 strains of Enterobacter cloacae were isolated from the inpatients between September, 2003 and November, 2004. 5 kinds of 16S rRNA methylase gene (including armA, rmtA, rmtB, rmtC and rmtD) and 9 kinds of AMEs gene [including aac (3)-I, aac(3)-II, aac(3)-III, aac(3)-IV, aac(6')-Ib, aac(6')-II, ant(3")-I, ant(2")-I and aph(3')-VI] were analyzed by PCR and verificated by DNA sequencing. RESULTS: In 40 strains of Enterobacter cloacae, the positive rates of genes of rmtB, aac(3)-II, aac(6')-I b, ant(3")-I, ant(2")-I and aph(3')-VI were 12.5% (5/40), 27.5% (11/40), 72.5% (29/40), 32.5% (13/40), 5.0% (2/40) and 5.0% (2/40), respectively. 8 kinds of the rest of genes were all tested negative. The total positive rate of AMEs gene was 85.0% (34/40). Among 29 strains of Enterobacter cloacae that the aac (6')-I b gene was positive, through PCR and verification by DNA sequencing, 7 strains (24.1%) were confirmed to take the aac(6')-I b-cr (the GenBank register number: EF375620, EU159121) alone, 18 strains (62.1%) were confirmed to take the aac(6')-I b-Suzhou (EU085533) alone, 3 strains (10.3%) were confirmed to take both aac(6')-I b-Suzhou and aac(6')-I b-cr while only 1 (3.4%) was aac(6')-I b (the classical type). CONCLUSION: There was lower positive rate of 16S rRNA methylase gene but very high AMEs genotypes in Enterobacter cloacae isolated from inpatients and the finding of rmtB gene was reported for the first time in the world. At least 5 kinds of AMEs gene existed in Enterobacter cloacae were isolated and they were the new host of both gene of aac(6')-I b- cr and aac(6')-I b-Suzhou, with aac(6')-I b-Suzhou gene was the predominance subtype in aac(6')-I b.